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The Dynamics of DNA in Salmonella Persisters in Macrophages

Award Number: F31AI176589
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 05/01/2023
PERIOD OF PERFORMANCE END DATE: 05/29/2025

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The Dynamics of DNA in Salmonella Persisters in Macrophages - Abstract Bacterial persistence contributes to antibiotic treatment failure and the relapse of many recalcitrant infections. Persisters are a subpopulation of transiently non-growing bacteria capable of surviving antimicrobial attacks from antibiotics and the immune system and eventually resuming growth. Many pathogens including, Salmonella enterica, Mycobacterium tuberculosis, and Staphylococcus aureus, form persisters within macrophages where they survive extended periods of time. It was shown that, although non-growing, Salmonella persisters retain the ability to express and inject effector proteins in macrophages leading to interference with the host immune response and supporting persister survival. Nonetheless, persisters remain vulnerable to macrophage-induced DNA damage in the form of double stranded breaks (DSBs) and require DSB repair through homologous recombination. Strikingly, intramacrophage Salmonella persisters also actively replicate chromosomal DNA and can accumulate more than four chromosome equivalents of DNA. I have found that persisters replicate complete chromosomes despite growth arrest and that this chromosome amplification is associated with a higher frequency of persister regrowth. I hypothesize that stresses encountered upon macrophage entry trigger a specific state of growth arrest where atypical chromosome replication is enabled and then favors repair of chromosomal DSBs by homologous recombination. To evaluate this hypothesis, I will decipher the mechanisms and consequences of DNA synthesis in Salmonella persisters. In Aim 1, I will characterize the contribution of chromosome amplification to homologous recombination and thus persister survival. I will use gene conversion assays to measure homologous recombination in persisters with high DNA content (1.1). I will then assess how DSB repair affects persister regrowth by tracking DSB repair using fluorescent imaging and transcriptional reporters of the DNA damage response (1.2). In Aim 2, I will determine the basis for DNA synthesis despite growth arrest including the requirements for initiation of DNA synthesis and intramacrophage conditions that trigger this atypical DNA synthesis. I will determine the requirements for initiation of chromosome replication at oriC through minichromosome replication assays (2.1). I will assess the macrophage triggers for atypical DNA replication by evaluating DNA accumulation of persisters in genetically- altered macrophages (2.2). Altogether, this research will further our understanding of intracellular persisters including formation, maintenance of the persistent state, and re-growth. Mechanistic understanding of persister survival will ultimately contribute to the development of approaches for targeting persisters, enhancing antibiotic efficacy, and preventing the development of antibiotic resistance.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $8,609 )
2025	2025	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	001	3	6/24/2025	NON-COMPETING CONTINUATION	-$28,247
2025	2025	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	3	4/18/2025	NON-COMPETING CONTINUATION	$36,856
														Subtotal = $8,609

Issue Date FY: 2024 ( Subtotal = $36,856 )
2024	2024	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	001	2	5/7/2024	NON-COMPETING CONTINUATION	$1,280
2024	2024	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	2	3/29/2024	NON-COMPETING CONTINUATION	$35,576
														Subtotal = $36,856

Issue Date FY: 2023 ( Subtotal = $41,676 )
2023	2023	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	1	3/6/2023	NEW	$41,676
2023	2023	PRESIDENT AND FELLOWS OF HARVARD COLLEGE	25 SHATTUCK ST	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	001	1	3/6/2023	NEW	$0
														Subtotal = $41,676

Grand Total All Awards = $87,141

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The Dynamics of DNA in Salmonella Persisters in Macrophages

Award Number: F31AI176589

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 05/01/2023

PERIOD OF PERFORMANCE END DATE: 05/29/2025

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